The KSR1 scaffold translocates from the cytosol to the plasma membrane upon Ras activation and coordinates the assembly of a large multiprotein complex that functions to regulate the intensity and duration of ERK cascade signaling. In the past fiscal year, we have identified a hydrophobic motif in the proline-rich sequence of MEK1 and MEK2 that is required for constitutive binding to the KSR1 scaffold and find that KSR1 forms a ternary complex with B-Raf and MEK in response to growth factor treatment that enhances B-Raf-mediated MEK activation. Strikingly, we have also found that docking of active ERK to the KSR1 scaffold allows ERK to phosphorylate KSR1 and B-Raf on feedback sites. Phosphorylation of the feedback sites attenuates ERK cascade signaling by promoting the dissociation of the B-RAF/KSR1/MEK complex and causing the release of KSR1 from the plasma membrane. This year our laboratory has also taken a proteomic approach to further investigate the functional properties of the two mammalian KSR scaffolds, KSR1 and KSR2. These studies have revealed that both KSR1 and KSR2 interact with the core kinase components of the ERK cascade and have a common function in promoting receptor tyrosine kinase-mediated ERK signaling. Remarkably, these studies also found that the protein phosphatase calcineurin selectively interacts with KSR2 and that KSR2 uniquely contributes to Ca2+-mediated ERK signaling. In response to increased Ca2+ levels, we find that calcineurin dephosphorylates KSR2 on specific sites and, as a a result, regulates the membrane-localization and scaffolding activity of KSR2. Moreover, our stuidies have shown that depletion of KSR2 impairs Ca2+-mediated ERK activation and signaling in two different cell lines that express KSR2, INS1 pancreatic beta-cells and NG108 neuroblastoma cells. These findings identify KSR2 as a Ca2+-regulated ERK scaffold and reveal a new mechanism whereby Ca2+ impacts Ras to ERK pathway signaling. Due to success of the proteomic approach in elucidating the function and regulation of the KSR scaffolds, we have expanded our use of these techniques to include the the mammalian CNK scaffold family, comprised of the CNK1, CNK2A, CNK2B and CNK3 proteins. Not surprising given the similar domain structure of the CNK family members, this analysis identified several common CNK-interacting proteins;however, it also revealed key differences in the CNK complexes that suggest important functional diversity. In particular, our studies revealed that the major binding partners of the CNK1 scaffold are members of the cytohesin family of Arf guanine nucleotide exchange factors and that the CNK1/cytohesin interaction is critical for activation of the PI3K/AKT cascade downstream of insulin and IGF-1 receptors. We have identified an 83 amino acid domain located in the C-terminal region of CNK1 that interacts constitutively with the coiled-coil domain of the cytohesins and find that CNK1 facilitates the membrane recruitment of cytohesin-2 following insulin stimulation. Moreover, through protein depletion and protein add-back experiments, we find that the CNK1/cytohesin interaction promotes signaling from plasma membrane-bound Arf GTPases to the PIP5Ks to generate a PIP2-rich microenvironment that is critical for both the membrane recruitment of IRS1 and signal transmission to the PI3K/AKT cascade. The insulin pathway is vital for energy metabolism and growth, and its dysregulation is a major contributor to human disease. These findings provide important new mechanistic insight regarding insulin pathway regulation and define a role for CNK1 as a regulator of cytohesin function and a positive modulator of insulin signaling.